The Study Of Preclinical Pharmocokinetics Of Humanized Monoclonal Anti-ricin Antibody(MIL50)

ObjectiveThe character of preclinical pharmacokinetics of humanized monoclonal anti-ricin an-tibody was studied and the study was taking a certain progress.We hope that this study can provide the clinical experience with theoretical basis and boosts the development of MIL50.MethodAn enzyme-linked immunosorbent assay（ELISA）was established and applied for the detection of concentration of MIL50 in rhesus monkey serum.The assay was strictly validated and it met the requirement of method which was ruled by FDA.In order to investigate the tissue distribution and excretion of MIL50 in Wistar rats,MIL50 was labeled with ¹²⁵I using the Iodogen method.Then the concentration of ¹²⁵I-MIL50 in serum and tissues、body fluids and excretions was measured at different time.Result1.The validation of enzyme-linked immunosorbent assay for the detection of rhesus monkey serum samplesIn this study,we have established an enzyme-linked immunosorbent assay to detect the concentration of MIL50 in rhesus monkey serum.The results shows that the linearity was good when the concentration of MIL50 ranges from 0.1¹0 ng·mL^-1,the lowest limit of quantification is 0.1 ng·mL^-1.The inter-precision and intra-precision of the method were4.2¹4.6%and 5.6¹7.9%respectively.The accuracy was 1.3⁹.9%。There was no dilution effect when the samples were diluted and the stability was very good when it was stored at-20℃for 5months.The method was highly meet the methodology requirement that FDA ruled.2.The pharmacokinetics study of MIL50 in rhesusThe half-lives of MIL50 at different doses(1.0,4.0,16mg·kg^-1)were 19.1±0.7、15.8±2.5and 16.7±3.9 days respectively when administrated intravenously.There was no significant difference between the three groups.The AUC_last were 479034±36792、2640882±524576and 13461992±2170727 day?ng?mL^-1 respectively,Therefor the ratio was 1:5.5:28.1.The clearance rate was 2.10±0.15、1.56±0.29、1.21±0.18 mL?day^-1?kg^-1.The apparent volume of distribution were 57.55±2.75、35.21±6.71、28.86±7.03mL?kg^-1.The parameters of the last two groups had no significant differences.To sum up,the pharma-cokinetics of MIL50 was linear kinetics within the dose of 1.0¹6 mg·kg^-13.The pharmacokinetics of MIL50 in Wistar ratsTo examine the effect of Ricin on the MIL50,we have practice the following experience.One group of Wistar rats were administrated ¹²⁵I-MIL50 intravenously at a dose of 1 mg·kg^-1after an intraperitoneal injection of Ricin at a dose of 12 ug·kg^-1.Another group of rats were administrated ¹²⁵I-MIL50 intravenously at the same dose directly as the control group.Then the serum samples were collected at different times.The result shows that ¹²⁵I-MIL50 was eliminated faster after 14 days in the Ricin administrated group.Ricin could fasten the elimination of ¹²⁵I-MIL50 when the concentration of ¹²⁵I-MIL50 was low in Wistar rats.The main pharmacokinetics parameters of ¹²⁵I-MIL50such as AUClast、apparent volume of distribution、clearance rate、half life、MRTlast in the two groups had no significant differences.That reminds us that Ricin had no effect on MIL50 within 14 days and it might because that the antibody was far more than Ricin and the conjugation of Ricin-MIL50 was eliminated with the ¹²⁵I-MIL50that unconjugated.However,there were significant in main parameters within 34 days and it might because of the interaction between antigen and antibody.4.The distribution of ¹²⁵I-MIL50 in Wistat ratsTo evaluate the character of distribution of ¹²⁵I-MIL50 in Wistar rats after administrat-ed at a dose of 2.2mg·kg^-1,we have preform the experiment of tissues distribution.After administration of MIL50,the male rats were executed at 1h、24h、8d、35d and the female rats were executed at 24h,each group contain 6 rats and then various tissues、organs and biofluids were collected.Then the tissues were weighted and cut into pieces.The protein in sample was precipitated by TCA,then centrifuged and determined the total and the pr-ecipitated radioactive concentration.The result showed that ¹²⁵I-MIL50 was detected in all tissues and fluids which demonstrated that MIL50 could distributed in every organs and tissues.The concentration of ¹²⁵I-MIL50 in serum was always higher than that in other tissues and this means MIL50 can not specificly conjugated with tissues and there was no accumulation in tissues.The radioactive in stomach contents might because the fragment of MIL50 after it was metabolism by enzyme.The high concentration of ¹²⁵I-MIL50 in kidney and bladder suggested ¹²⁵I-MIL50 might eliminated through urine and this should be tested in the next experients.In addition,the concentration in the brain,spinal fat was very low,predicting that ¹²⁵I-MIL50 is not easy to enter the blood brain barrier and the lipophilic tissues.After the T test we found that there was no sexual difference of MIL50 in tissues.5.The excretion of ¹²⁵I-MIL50 in Wistar ratsTo study the character of excretion of MIL50 in Wistar rats,we collected the urine、bile and faecalis and detected the radioactivity at different time and then calculated the total radioactivity.As a result,the cumulative excretion rate of ¹²⁵I-MIL50 was 62.6%in urine,and 15.5%in feces within 27days and 4.5±1.6%in bile within 5 days.Urinary excret-ion represented the major pathway of elimination of ¹²⁵I-MIL50.Conclusion1.The ELISA method we have established was highly meet the methodology require-ment that FDA ruled and can be used to detect the concentration of MIL50 samples.2.The result of pharmacokinetics of MIL50 in rhesus shows that MIL50 was linear kinetics within the dose of 1.0¹6 mg·kg^-1in rhesus.3.The result of pharmacokinetics of MIL50 in Wistar rats shows that ¹²⁵I-MIL50 was eliminated faster after 14 days in the Ricin administrated group,it might because of the interaction between antigen and antibody.4.The result of distribution of MIL50 shows that it can widely distributed in tissues and can not specificly conjugated with tissues.It is not easy for MIL50 to enter the blood brain barrier and the lipophilic tissues.5.The result of excretion of ¹²⁵I-MIL50 in Wistar rats shows that Urinary excret-ion represented the major pathway of elimination of ¹²⁵I-MIL50.